WO2001060151A1 - Mdel rats with the onset of prostatic cancer - Google Patents
Mdel rats with the onset of prostatic cancer Download PDFInfo
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- WO2001060151A1 WO2001060151A1 PCT/JP2000/003825 JP0003825W WO0160151A1 WO 2001060151 A1 WO2001060151 A1 WO 2001060151A1 JP 0003825 W JP0003825 W JP 0003825W WO 0160151 A1 WO0160151 A1 WO 0160151A1
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- prostate cancer
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
- C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates to a model rat for the generation of prostate cancer that can generate prostate cancer including invasive cancer and can be stably passaged, and a method for establishing the same. More specifically, prostate cancer, including invasive cancer by the age of 15 weeks, into which the SV (simian virus) 40 T gene, which is under the control of the promoter of the probasin gene, has been introduced.
- the present invention relates to a model rat for the development of prostate cancer that can occur in almost all cases and can be stably passaged, particularly to the PBSVT transgenic rat line 2971, and a method for establishing the same.
- the present invention relates to a method for screening for a substance for promoting and / or progressing prostate cancer or a substance for suppressing the occurrence and / or progression of prostate cancer using the model rat for prostate cancer development.
- Prostate cancer is the second leading cause of cancer death in Western Europe. Such prostate cancer is currently the only cause of cancer death in Japan, but it is expected to increase rapidly with an increase in life expectancy. Although the etiology of prostate cancer is poorly understood, it is initially androgen-dependent and then independent, becoming resistant to therapeutic agents. In order to elucidate the mechanism of prostate cancer development through such multi-steps, various animal models and various strains of prostate cancer model animals are required.
- ACI ZS eg-lattice-Mouth-Isband-Lobund-Wistar-lattice requires a long latency period of about 20 months to create an animal model that produces a high frequency of spontaneous malignancies. It has been reported that it costs money.
- ACIZS eg-type rat prostate lateral lobe cancer is the most moderately differentiated non-metastatic adenocarcinoma and has a cribriform pattern. It is known that adenocarcinoma with a small number sometimes shows metastasis.
- N-methyl-N-ditrosorerea MNU
- N-ditrosobis (2-oxopropyl) amine BOP
- 3,2′-dimethyl-4-aminobiphenyl as carcinogens.
- a rat prostate cancer model was established using (DMAB) and 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridin (PhIP).
- DMAB 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridin
- Jpn. J. Cancer Res., 76: 803-808, 1985 The lesions of the prostate cancer originated in the ventral lobe and showed a largely noninvasive phloem pattern. Its size was generally small and free of metastases.
- prostate cancer development due to DMA B can be modified by long-term administration of high doses of testosterone propionate (TP).
- TP testosterone propionate
- Ki-r gene is involved in cancer development in prostate and testis by DMAB.
- Adenocarcinoma of the abdominal lobe 3 out of 9 cases, adenocarcinoma of the dorsal lobe 1 5 cases out of 3 cases, testicular adenocarcinoma 1 1 out of 1 case showed a point mutation in the Ki-ras gene (point-to-point No mutations were found in the Ha-ras gene or the p53 gene (Mol. Carcinog., 13: 21-26, 1995).
- Atypical hyperplasia in all regions and abdominal lobe cancers is immunohistochemically positive for androgen receptor, but invasive carcinomas in the dorsal and anterior lobes are not, and invasiveness induced by DMAB and TP
- the growth of three cell lines established from dorsal lobe prostate cancer in vivo is androgen-independent (Jpn. J. Cancer Res., 87: 1218.1226, 1996, J Toxicol. Pathol., 11: 27-32, 1998).
- testicles are removed after treatment with DMAB alone or with DMAB and TP, the progression of androgen receptor-one-positive abdominal lobe cancer is completely stopped, but dorsal and anterior lobe prostates and testes formed in the prostate.
- prostate cancer In the induction of prostate cancer by the administration of carcinogens as described above, various types of prostate cancer can be generated, but since the development of cancer requires long-term animal breeding of one year or more, Recently, animal models incorporating cancer-causing genes have been actively developed.
- Several transgenic animals with prostate cancer have been reported to date.
- mice that incorporated the 11.5 kb promoter of Probasin and the SV40 large T antigen gene produced by Yan in 1999, developed prostate cancer.
- Mice that incorporated the gene for the C 3 (1) promoter and the SV40 large T antigen produced by Maroulakoou in 1994, developed prostate cancer.
- Mice that incorporated the C3 (1) promoter and the Polyoma middle T gene produced by Tehranian in 1996, developed prostate cancer.
- Zhang reported that mice with the C3 (1) promoter and the human be1-2 (human bcl-2) gene developed hyperplasia in the prostate but not cancer. Did not reach.
- Rats like mice, have a short pregnancy and sexual maturation period, have about 10 litters, and weigh about 10 times that of mice, so that sufficient materials are available for analysis. In addition, there are many useful points such as the accumulation of data on many chemical carcinogenesis.
- An object of the present invention is to provide a model rat for prostate cancer development that can generate prostate cancer including invasive cancer and can be stably subcultured. Disclosure of the invention
- the present inventors have intensively studied to solve the above-mentioned problems, and have studied a transgenic rat obtained by introducing a gene for SV40 large T antigen under the control of the promoter of the probasin gene into a rat.
- a transgenic rat obtained by introducing a gene for SV40 large T antigen under the control of the promoter of the probasin gene into a rat.
- a model rat of prostate cancer occurrence that can be used can be established, and have completed the present invention. That is, the present invention provides a model rat for prostate cancer development (Claim 1) that can generate prostate cancer including invasive cancer and can be stably subcultured, and under the control of the promoter of the probasin gene.
- the gene for the SV40 large T antigen is introduced into the model rat for prostate cancer development according to claim 1 (claim 2), or the probasin gene promoter of the rat cut with EcoRI and Ncol is 5'- Downstream of the flanking region (1 426 to 1332 458 bp), the gene for SV40 large T antigen (5164 to 2676) cut out by Ncol and BamHI is linked.
- the 2944 bp PBSVT transgene obtained by the above procedure was introduced into fertilized eggs of Sprague-Dawley rats, and the fertilized eggs after introduction were transplanted into foster parents.
- the transgenic male rat obtained from the foster parent is wild-type Sprague-Dawley. 3.
- 971 A model rat for the development of prostate cancer according to any one of claims 1 to 3, which is a one-line system (claim 4).
- the present invention provides a promoter for the probasin gene of a rat cut with EcoRI and Ncol, which is located at the 5'-flanking region (-425-bp 32-458 bp) of the promoter.
- a PBSVT transgene consisting of 2944 bp obtained by binding the SV40 large T antigen gene (5164-26676) cut out with BamHI was added to the plasmid.
- -(Sprague-Dawley) rats are introduced into a fertilized egg, the fertilized eggs after the introduction are transplanted into a foster parent, and the transgenic rat obtained from the foster parent is used as a wild-type Sprague-Dawley-type rat.
- a transgenic rat that produces prostate cancer is selected.
- a method for establishing a model rat for the development of prostate cancer that can generate stable prostate cancer and that can be stably passaged (Claim 5), and a method for generating prostate cancer including invasive cancer.
- the prostate cancer generation model including the invasive cancer according to claim 5, wherein the model rat model for prostate cancer generation that can be stably subcultured is the PBSVT transgenic rat line 2971.
- the present invention relates to a method for establishing a model rat for the development of prostate cancer that can be stably subcultured.
- the present invention provides a method for administering a test substance to a model rat for prostate cancer development according to any one of claims 1 to 4 before and after the onset of prostate cancer, wherein the degree of the onset, Z or progression of prostate cancer in the model rat is measured.
- the prostate cancer development and Z or progression promoting substance according to claim 7, wherein the measurement and evaluation of the degree is analysis and evaluation of a histopathological image of prostate cancer obtained from a model rat of prostate cancer development.
- the method for screening for substances inhibiting the onset and progression of Z or progression (Claim 8) and the measurement and evaluation of the degree of onset and progression of prostate cancer and the progression of Z or progression are performed by prostasis produced in prostate cancer cells 8.
- PAP acid phosphatase
- PSA prostate specific antigen
- the measured value of a wild-type rat of the same type as the model rat of prostate cancer development is used in the screening method for a progression inhibitor (Claim 9).
- a method for screening prostate cancer occurrence and Z or progression promoting substances or occurrence and / or progression inhibiting substances (claim 10) according to any one of claims 7 to 9, characterized in that they are compared and evaluated.
- the prostate cancer development and Z or progression inhibiting substance obtained by the screening method for prostate cancer development and Z or progression promoting substance or development and / or progression inhibiting substance according to any one of claims 0 to 12.
- FIG. 1 is a diagram showing a PVSVT gene fragment used for creating a prostate cancer transgenic rat model of the present invention.
- the model rat of the present invention for the development of prostate cancer is particularly limited as long as it is a rat line that can generate prostate cancer including invasive cancer in almost all cases and can be passaged stably. Although not specifically regulated, it binds directly to p53 and Pb proteins under the control of the promoter of the probasin gene, a gene that is specifically expressed in the prostate and encodes a protein regulated by androgens and zinc. Induces cancer by inactivating functions. From a transgenic rat into which a transgene (PBSVT transgene) linked to the gene for SV40 large T antigen is introduced. An established model rat for the development of prostate cancer, for example, the PBS VT transgenic rat 297L strain can be specifically mentioned.
- PBSVT transgene transgene linked to the gene for SV40 large T antigen
- a method for establishing a model rat for the development of prostate cancer As a method for establishing a model rat for the development of prostate cancer according to the present invention, a method using a known method for producing a transgenic animal (for example, Proc. Natl. Acad. Sci. USA 77: 7380-7384, 1980) is used.
- a gene fragment in which the SV40 large T antigen gene is located under the control of the above-mentioned probasin promoter is used as a transgene will be described as an example.
- a PBS VT transgene prepared according to a known method is put into an appropriate expression vector and amplified. After amplification, a gene fragment (PBS VT transgene) is cut out, and the PBS VT transgene is microinjected.
- a model rat line for the development of prostate cancer can be established by observing the pathological tissue of the prostate and selecting a transgenic rat that develops prostate cancer.
- a promoter derived from a prostate specific antigen (PSA) or a prostate specific membrane antigen (PSMA) which is specifically expressed in the prostate can be used.
- the gene for the SV40 large T antigen that induces cancer the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40,
- the E1A gene of an influenza virus and the HPV16 gene of a human papilla virus can be used.
- an electrostatic pulse method, a ribosome method, a calcium phosphate method, or the like can be used in place of the above-mentioned mouth opening method.
- the PBSVT transgenic rat strain 2971 which is a model rat for the development of prostate cancer according to the present invention, is a 5′-flanking promoter of the rat probasin gene promoter cut with EcoRI and Ncol. Downstream of the region (1 4 2 6 to +3 2 4 5 8 13), the SV40 large T antigen gene (5 16 4 to 2 6 7 6) cut by Ncol and BamHI The resulting 2944 bp PBS VT transgene is introduced into fertilized eggs of a Sprague-Dawley rat, the fertilized eggs after the introduction are transplanted into a foster parent, and the transfection obtained from the foster parent is performed.
- the transgenic rat is crossed with a wild-type Sprague-Dawley rat, and the resulting transgenic rat is similarly passaged thereafter.
- the invasive cancer can be generated including prostate cancer, which has been established as Moderura' capital of prostate cancer occurrence can be stably passaged.
- This PBSVT VT transgenic rat line 29771 has been passed on at the No. 1 Department of Pathology, Faculty of Medicine, Nagoya Ritsumeikan University, and under certain conditions, interested parties can receive a subdivision.
- the model rat for prostate cancer development can be used for elucidating the mechanism of prostate cancer development and for screening a substance that promotes or suppresses the development and progression of prostate cancer.
- a test substance is administered to a model rat of prostate cancer development of the present invention before or after prostate cancer development, and the occurrence of prostate cancer and the degree of Z or progress in the model rat are determined.
- Measurement and evaluation methods can be mentioned.
- examples of the method for measuring and evaluating the degree of prostate cancer occurrence and Z or progression include a method for analyzing and evaluating a pathological image of prostate cancer obtained from a model rat to which a test substance has been administered.
- the substance that suppresses prostate cancer occurrence and Z or progression obtained by the screening method of the present invention may be used as a prophylactic, suppressive, or therapeutic agent for prostate cancer.
- administration of a test substance before the onset of prostate cancer is suitable for screening for a prophylactic cancer preventive agent
- administration of a test substance after the onset of prostate cancer is suitable for treatment of a prostate cancer therapeutic agent ⁇ Suitable for screening for symptom improvers.
- a substance that promotes the development of prostate cancer and promotes Z or progression is considered to be useful when it is desired to sufficiently generate prostate cancer in a model animal in which the development of prostate cancer is insufficient.
- composition of the present invention used for treating a patient in need of suppressing the occurrence and progression of prostate cancer or the prostate cancer is characterized by comprising a substance inhibiting prostate cancer development and / or Z or progression as an active ingredient. Contains known substances required for
- pBluescriptllKS As the gene for the SV40 large T antigen, a gene integrated into a multicloning site of pBluescriptllKS (-) as an Ncol-BamHI fragment, provided by the Human Science Research Resource Bank was used. From this pBluescriptllKS (-), the gene for SV40 large T antigen (5164 to 2676) cut with Ncol and BamHI was cut with EcoRI and Ncol. By binding to the downstream of the 5'-flanking region of the P-rat probasin gene promoter (-428 to 132, 458 bp), a PBS VT transgene was constructed (Fig. 1).
- the PBS VT transgene capable of expressing both the SV440 large T antigen and the small t antigen consisting of 294 bp was amplified using plasmid pBluescriptll (manufactured by Transgene). After digestion with the restriction enzymes EcoRI and BamHI, 1% agarose gel electrophoresis was performed to remove the plasmid portion and separated as a linear DNA fragment. The separated DNA fragments were recovered using GENECLEANE II (BIO101 INC), and the DNA obtained after purification was adjusted to 5 fi / m1 with an injection buffer (10 mM Tris-containing O.lmM EDTA). HCl, pH 7.6) and stored at 120 ° C until used for injection.
- an injection buffer (10 mM Tris-containing O.lmM EDTA).
- Microinjection of the PBS VT transgene solution into the pronuclear stage fertilized eggs of the rat was performed as follows.
- An 8-week-old Sprague-Dawley (Sprague-Dawley) female rat was subjected to a 12-hour light-dark cycle (bright time 4: 00-: L 6: 00), temperature about 23 ° C, humidity about
- the animals were bred at 55% and the hormonal cycle of the female was observed by the vaginal smear method, and the date of hormone treatment was selected.
- Superovulation was performed by intraperitoneally administering 150 IU kg of pregnant female serum gonadotropin (PMS Zenyaku, manufactured by Nippon Zenyaku Co., Ltd.) to female rats, and 48 hours later, 75 IUZ kg of human chorionic gonadotropin (Puberogen, manufactured by Sankyo Organ Co., Ltd.) was administered, and then mated with a male. The pronuclear stage fertilized eggs were collected by the method.
- mK RB solution Y. Yoshida and MC Chang, J. Reprod. Fertil., Vol. 36, pp9-22 (l974) was used.
- the collected fertilized eggs were subjected to enzymatic treatment at 37 ° C for 5 minutes in mKR B solution containing 0.1% hyaluronidase (Sigma) to remove cumulus cells.
- the enzyme was removed by washing 3 times with solution B, to DNA injection operation C 0 2 incubator base - the coater (C_ ⁇ 2 5%, 3 7:, saturated humidity) and stored in.
- the PBS VT transgene solution was introduced into the male pronucleus of the fertilized eggs of Wistar rat thus prepared so that the number of molecules per fertilized egg was 10 molecules.
- the introduced 200 fertilized eggs were transplanted into 10 foster parents to obtain 40 offspring.
- the first male transgenic rat was crossed with a healthy wild-type SD female rat to produce the first hybrid (F1), and the lesions in systemic organs were examined over time.
- Line 29771 was able to be stably passaged and established as a PBS VT transgenic rat.
- the occurrence of hydrocephalus increased with the passage of time, and the strain was interrupted.
- F0 of the 2944 strain and the 2971 strain developed bilateral lower limb paralysis from around the age of 12 weeks and died at 14 and 25 weeks. In both cases, diffuse atypical hyperplasia was observed in the ventral lobe of the prostate, but no cancer lesion was observed.
- the SV40 T antigen was expressed on all lobes of the prostate gland, but was highly expressed especially on the abdominal lobe. In both cases, spinal cord tumors were observed, and in addition, sublingual tumors were observed in the 2971 line, and both were malignant small round cell tumors and were positive for SV40 T antigen.
- Table 1 Overall findings of the PBS VT transgenic rat line 2971 '' for the deaths and slaughter at passages 2971 and 2944 (In case of death or slaughter) "and Table 2" Overall findings of PBSVT transgenic rat line 2944 (in case of death or slaughter) ".
- histopathological findings of those prostates were It is shown in the table “Histopathological findings of the prostate in PBS VT transgenic rats”. As shown in Table 1, it was confirmed that the 2971 rat could be subcultured. Except for the prostate, small cell undifferentiated malignant tumors were found around the tongue and spinal cord. In addition, as shown in Table 2, many individuals in the 2944 series rat died of hydrocephalus. Apart from the prostate, small cell undifferentiated malignant tumors were found in the tongue, around the spinal cord, and elsewhere. table 1
- Male PBSVT transgenic rats were divided into three groups at the age of 5 weeks, the first group was used as a control group, the second group was orchidectomized, and the third group was testosterone filled in a 1.5 cm silicon tube. Subcutaneously transplanted, all groups were sacrificed at the age of 15 weeks by autopsy, and prostate lesions were examined histopathologically. In the first group of controls, all patients had diffuse high-grade atypical hyperplasia in the anterior lobe of the prostate gland, and some developed cancer. In the second group, the prostate was completely atrophied and no neoplastic lesions were observed. In group 3, the entire prostate was markedly swollen, and the incidence of invasive cancer was high.
- This animal model can cause prostate cancer early and at a high rate. Therefore, it is extremely useful as a prostate cancer model for analyzing the prostate carcinogenesis process in detail and developing therapeutic agents for prostate cancer.
- the transgenic rat of the present invention is useful in the medical research field due to its trait.
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Abstract
Model rats with the onset of prostatic cancer in which prostatic cancer including infiltrating cancer can be induced and which can be subcultured in a stable state. SV40 large T antigen gene is ligated to the downstream of rat probasin gene promoter and the thus obtained PBSVT transgene is transferred into a fertilized ovum of a Sprague-Dawley rat. After the transfer, the fertilized ovum is transplanted into a host. A transgenic rat obtained from the host is then mated with a wild Sprague-Dawley rat and the transgenic rats thus obtained are similarly subcultured thereafter. Transgenic rats with the onset of prostatic cancer are selected by observing pathological prostatic tissues, thereby establishing model rats with the onset of prostatic cancer in which prostatic cancer including infiltrating cancer can be induced and which can be subcultured in a stable state.
Description
明 細 前立腺癌発生モデリレラッ 卜 技術分野 Details Modeling of prostate cancer development
本発明は、 浸潤癌を含む前立腺癌を発生することができ、 安定的に継 代することができる前立腺癌発生のモデルラッ ト及びその樹立方法に関 する。 より詳しくは、 プロバシン遺伝子のプロモ一夕一の制御下にある S V (シミアンウィルス、 simian virus) 4 0ラ一ジ T抗原遺伝子が導 入された、 1 5週齢までに浸潤癌を含む前立腺癌をほぼ全例に発生する ことができ、 安定的に継代することができる前立腺癌発生のモデルラッ ト、 特に P B S V T トランスジエニックラッ ト 2 9 7 1系統や、 その樹 立方法に関する。 また本発明は、 かかる前立腺癌発生のモデルラッ トを 用いた前立腺癌の発生及びノ若しくは進行促進物質又は発生及び Ζ若し くは進行抑制物質のスクリーニング方法等に関する。 背景技術 The present invention relates to a model rat for the generation of prostate cancer that can generate prostate cancer including invasive cancer and can be stably passaged, and a method for establishing the same. More specifically, prostate cancer, including invasive cancer by the age of 15 weeks, into which the SV (simian virus) 40 T gene, which is under the control of the promoter of the probasin gene, has been introduced. The present invention relates to a model rat for the development of prostate cancer that can occur in almost all cases and can be stably passaged, particularly to the PBSVT transgenic rat line 2971, and a method for establishing the same. In addition, the present invention relates to a method for screening for a substance for promoting and / or progressing prostate cancer or a substance for suppressing the occurrence and / or progression of prostate cancer using the model rat for prostate cancer development. Background art
西欧において前立腺癌は癌死亡の第 2番目の原因となっている。 かか る前立腺癌は、 日本において現時点では癌死亡の 1 1番目の原因でしか ないが、 平均寿命の延長に伴い、 急激に増加することが予想されている。 前立腺癌の病因は殆ど解明されていないが、 初期段階ではアンドロゲン 依存性であり、 その後非依存性となり、 治療薬に対して抵抗性を有する ようになる。 このように多段階を経由する前立腺癌発生のメカニズムを 解明するには、 色々な動物の、 かつ種々の系統の前立腺癌モデル動物が 必要とされている。 Prostate cancer is the second leading cause of cancer death in Western Europe. Such prostate cancer is currently the only cause of cancer death in Japan, but it is expected to increase rapidly with an increase in life expectancy. Although the etiology of prostate cancer is poorly understood, it is initially androgen-dependent and then independent, becoming resistant to therapeutic agents. In order to elucidate the mechanism of prostate cancer development through such multi-steps, various animal models and various strains of prostate cancer model animals are required.
前立腺癌のいくつかの動物モデルがこれまでに作製され、 前立腺癌発
0/0 5 生のメカニズムやその修飾因子を解明するために用いられてきた。 A C I ZS e g系ラッ トゃ口一バンド—ウイス夕一（Lobund- Wistar)系ラッ トで自然発生的な悪性腫瘍を高い頻度で発生する動物モデルを作るには 約 2 0ヶ月という長い潜在期を要することが報告されている。 A C I Z S e g系ラッ 卜の前立腺側葉癌は最も適度に分化された非転移性の腺癌 であり篩状のパターンをしており、 またローバンドーゥイス夕一系ラッ トの前立腺癌は、 分化の少ない腺癌で時々転移性を示すことが知られて いる。 Several animal models of prostate cancer have been generated to date 0/0 5 It has been used to elucidate the mechanism of production and its modifiers. The ACI ZS eg-lattice-Mouth-Isband-Lobund-Wistar-lattice requires a long latency period of about 20 months to create an animal model that produces a high frequency of spontaneous malignancies. It has been reported that it costs money. ACIZS eg-type rat prostate lateral lobe cancer is the most moderately differentiated non-metastatic adenocarcinoma and has a cribriform pattern. It is known that adenocarcinoma with a small number sometimes shows metastasis.
これまでに本発明者らは、 発癌物質として、 N—メチル—N—二トロ ソゥレア （MNU)、 N—二トロソビス （ 2—ォキソプロピル） ァミン （B O P)、 3， 2 ' —ジメチルー 4一アミノ ビフエニル （DMAB) 及び 2 —アミノー 1 一メチルー 6—フエ二ルイミダゾ [4 , 5— b]ピリヂン（P h I P) を用いて、 ラッ ト前立腺癌モデルを確立した。 発癌性物質の投 与と細胞増殖を組み合わせることによって、 前立腺癌を高い頻度で発生 するラッ 卜の作製に成功した（Jpn. J. Cancer Res. ,76:803-808， 1985)。 その前立腺癌の病巣は腹葉 (ventral lobe)に発生し、 大部分が浸潤性のな い篩状パターンを示した。 その大きさは、 一般に小さく転移も無かった。 To date, the present inventors have reported that N-methyl-N-ditrosorerea (MNU), N-ditrosobis (2-oxopropyl) amine (BOP), 3,2′-dimethyl-4-aminobiphenyl as carcinogens. A rat prostate cancer model was established using (DMAB) and 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridin (PhIP). By combining carcinogen administration and cell proliferation, we succeeded in producing a rat that produces prostate cancer at a high frequency (Jpn. J. Cancer Res., 76: 803-808, 1985). The lesions of the prostate cancer originated in the ventral lobe and showed a largely noninvasive phloem pattern. Its size was generally small and free of metastases.
DM A Bで誘発する前立腺癌の場合には、動物種による特異性があった。 すなわち、 F 3 44及び A C I 系ラッ 卜は最も感受性が高く、 ウイス夕 —(Wistar)系ゃスプラーグ—ダウレイ（Sprague-Dawley)系は抵抗性を 有していた。 しかし、 組織病理学的なパターンには明らかな種特異性は なかった。 In the case of DMAB-induced prostate cancer, there was species specificity. That is, the F344 and ACI-type rats were the most sensitive, and the Wistar- ゃ グ Sprague-Dawley-type had resistance. However, there was no apparent species specificity in histopathological patterns.
MNU及び B〇 Pと同様に、 DMA Bによる前立腺癌発生は、 テス ト ステロンプロピオネー ト （T P) を高用量で長時間投与して修飾するこ とができる。 本発明者らは、 T Pをシリコンチューブに入れて皮下に埋 め込み、 血清中のテス トステロン濃度を 1 0倍に高めたところ、 DMA
T/JP00/03825 Like MNU and B〇P, prostate cancer development due to DMA B can be modified by long-term administration of high doses of testosterone propionate (TP). The present inventors found that TP was implanted subcutaneously in a silicon tube and the testosterone concentration in serum was increased 10-fold. T / JP00 / 03825
Bの投与を受けたラッ トの生殖器における癌スペク トルが、 腹葉から背 側葉及び前葉ならびに精巣に移行することを見い出している（Cancer Res. ,51:1264-1269, 1991)。 腹葉の前立腺癌に比べて、 後者の部位に発生 した前立腺癌は浸潤的に成長する腺癌であり、 転移病巣は、 腹腔、 肝臓、 肺などに見られた。 腹葉から背側葉及び前葉ならびに精巣への移行やそ の浸潤性の程度は T Pの投与量と投与期間によって変わることも知られ てレ る (Cancer Lett.，83:11-116, 1994, Jpn. J. Cancer Res. ,86:645· 648(1995))。 The cancer spectrum in the genital tract of rats receiving B has been found to migrate from the abdominal lobe to the dorsal and anterior lobes and the testis (Cancer Res., 51: 1264-1269, 1991). Compared to prostate cancer of the abdominal lobe, prostate cancer that occurred in the latter site was an invasive growing adenocarcinoma, and metastatic lesions were found in the abdominal cavity, liver, lungs, and the like. It is also known that the transition from the abdominal lobe to the dorsal lobe, the anterior lobe, and the testes, and the degree of their invasiveness, vary depending on the dose and duration of TP (Cancer Lett., 83: 11-116, 1994, Jpn. J. Cancer Res., 86: 645 · 648 (1995)).
発癌性物質の投与により誘発された癌を分子的に分析した結果、 DM ABによる前立腺及び精巣における癌発生には、 Ki-r 遺伝子が関与し ていると考えられている。 腹葉の腺癌 9例中 3例、 側背葉の腺癌 1 3例 中の 5例、精巣の腺癌 1 1例中の 1例に、 Ki-ras遺伝子の点突然変異（ポ イントミユ ーテ一シヨン） が見られ、 Ha-ras遺伝子や p 5 3遺伝子には 変異はなかった（Mol. Carcinog., 13:21-26,1995)。 浸潤性と非浸潤性とを 問わず、 ras 遺伝子の突然変異に変わりは無く、 実験的なラッ トの前立 腺癌にお いては、 点突然変異に よ る ras プロ 卜 腫瘍遺伝子 (protooncogene)の活性化は癌の進行と結びついておらず、 また p 5 3遺 伝子の非活性化も進行とは関係がないと考えられている。 As a result of molecular analysis of cancer induced by administration of a carcinogen, it is considered that the Ki-r gene is involved in cancer development in prostate and testis by DMAB. Adenocarcinoma of the abdominal lobe 3 out of 9 cases, adenocarcinoma of the dorsal lobe 1 5 cases out of 3 cases, testicular adenocarcinoma 1 1 out of 1 case showed a point mutation in the Ki-ras gene (point-to-point No mutations were found in the Ha-ras gene or the p53 gene (Mol. Carcinog., 13: 21-26, 1995). Regardless of whether invasive or non-invasive, mutations in the ras gene remain the same, and in experimental rat prostate adenocarcinoma, the ras proto-oncogene (protooncogene) by point mutation Activation is not linked to cancer progression, and deactivation of the p53 gene is thought to be unrelated to progression.
全ての領域及び腹葉癌における異型過形成は、 免疫組織化学的にみて アンドロゲン受容体一陽性であるが、 背側葉及び前葉の浸潤性癌はそう ではなく、 DMABと T Pで誘発した浸潤性の背側葉前立腺癌から確立 した 3つのセルラインの生体中における生育はアンドロゲン非依存性で あること力 S幸 告されてレ る（Jpn. J. Cancer Res., 87:1218.1226, 1996, J. Toxicol. Pathol., 11:27-32, 1998) 。 D M A B単独又は D M A Bと T Pに よって処理した後睾丸を摘出すればァンドロゲン受容体一陽性の腹葉癌 の進行は完全に止まるが、 背側葉及び前葉前立腺や精巣に出来たアンド
ロゲン受容体—陰性の浸潤性癌の進行は止まらなかった（Jpn. J. Cancer Res.，90:23-30， 1999)。 腺癌のセルラインを用いたアンドロゲン受容体— 遺伝子の最近の研究結果は、 受容体のプロモーター領域における過メチ ル化（hypermethylation)を示しており、 これが、 浸潤性癌がアンドロゲ ン非依存性である理由であると考えられる。 Atypical hyperplasia in all regions and abdominal lobe cancers is immunohistochemically positive for androgen receptor, but invasive carcinomas in the dorsal and anterior lobes are not, and invasiveness induced by DMAB and TP The growth of three cell lines established from dorsal lobe prostate cancer in vivo is androgen-independent (Jpn. J. Cancer Res., 87: 1218.1226, 1996, J Toxicol. Pathol., 11: 27-32, 1998). When testicles are removed after treatment with DMAB alone or with DMAB and TP, the progression of androgen receptor-one-positive abdominal lobe cancer is completely stopped, but dorsal and anterior lobe prostates and testes formed in the prostate. The progression of the rogen receptor-negative invasive cancer did not stop (Jpn. J. Cancer Res., 90: 23-30, 1999). Recent studies of the androgen receptor-gene using adenocarcinoma cell lines have shown hypermethylation in the promoter region of the receptor, which indicates that invasive carcinomas are androgen-independent. This may be for some reason.
以上のような発癌性物質投与による前立腺癌の誘発においては、 種々 の前立腺癌を発生させることができるものの、 癌の発生には 1年以上の 長期間の動物の飼育を必要とすることから、 最近は、 癌発生性の遺伝子 を組み込んだ動物モデルが盛んに作られるようになってきている。 前立 腺癌のトランスジェニック動物もこれまでにいくつか報告されている。 1 9 9 5年にグリーンベルグ（Greenberg)が作製した、 プロノ シン (Probasin)の 0 . 5 k bのプロモーターと S V 4 0ラージ T抗原遺伝子 を組み込んだマウスは、 前立腺に癌を発生しその癌は転移性であつた。 1 9 9 6年にバリオス（Barrios)が作製した、プロバシン（Probasin)の 0 . 5 k bのプロモーターと ras T 2 4遺伝子を組み込んだマウスの前立腺 には過形成が見られたが癌は発生しなかった。 1 9 9 7年にヤン（Yan) が作製した、 プロバシン（Probasin)の 1 1 . 5 k bのプロモ一夕一と S V 4 0ラージ T抗原遺伝子を組み込んだマウスは、前立腺癌を発生した。 1 9 9 4年にマロウラクー（Maroulakoou)が作製した、 C 3 ( 1 ) プロ モ夕一と S V 4 0ラージ T抗原の遺伝子を組み込んだマウスは、 前立腺 癌を発生した。 1 9 9 6年にテーラ二アン（Tehranian)が作製した C 3 ( 1 ) プロモ夕一とポリォ一マミ ドル T (Polyoma middle T)の遺伝子を 組み込んだマウスは、 前立腺癌を発生した。 1 9 9 7年にッァン（Zhang) が C 3 ( 1 ) プロモタ一とヒト b e 1 - 2 (human bcl-2)の遺伝子を組み 込んだマウスは、 前立腺に過形成が発生したが癌には至らなかった。 1 9 9 6年にペレ—スターブル（Perez-Stable)が作製した、 胎盤 Gァ―グ
ロビン（f e t a l Gァ- g l ob i n)のプロモーターと S V 4 0ラ一ジ T抗原遺伝 子を組み込んだマウスは、 前立腺癌を発生した。 1 9 9 6年にキッッベ ルグ（Kitsberg)が作製した、 M M T Vのプロモーターと K G F遺伝子を 組み込んだものは前立腺に過形成を発生したが癌は発生しなかった。 癌の発生メカニズムを解明したり、その治療薬を開発するに当たって、 癌発生の動物モデルは無くてはならないものであり、 これまでに色々な 癌についてのモデル動物が作製されてきているが、 癌を誘発する物質、 方法、 動物種によって試験できる内容は異なるため、 できるだけ多くの 種類の動物モデルを確立することが望まれている。 またラッ トは、 マウ スと同様に妊娠期間、 性成熟期間が短く、 産仔数も 1 0匹前後と多く、 体重もマウスの 1 0倍程度あるため、 解析するのに十分な材料が得られ る点で優れており、 また、 多くの化学発癌に関するデータ一が蓄積して いるなど有用な点が多い。 しかし、 現在まで、 前立腺癌を確実に発生す ることができ、 安定的に継代することができる前立腺癌発生のモデルラ ッ トは確立されていなかった。 本発明の課題は、 浸潤癌を含む前立腺癌 を発生することができ、 安定的に継代することができる前立腺癌発生の モデルラッ トを提供することにある。 発明の開示 In the induction of prostate cancer by the administration of carcinogens as described above, various types of prostate cancer can be generated, but since the development of cancer requires long-term animal breeding of one year or more, Recently, animal models incorporating cancer-causing genes have been actively developed. Several transgenic animals with prostate cancer have been reported to date. A mouse that incorporated the 0.5 kb promoter of pronosin (Probasin) and the SV40 large T antigen gene, produced by Greenberg in 1995, developed cancer in the prostate gland, It was metastatic. Hyperplasia was observed in the prostate of a mouse, which was produced by Barrios in 1996 and incorporated the 0.5 kb promoter of Probasin and the rasT24 gene, but cancer occurred. Did not. Mice that incorporated the 11.5 kb promoter of Probasin and the SV40 large T antigen gene, produced by Yan in 1999, developed prostate cancer. Mice that incorporated the gene for the C 3 (1) promoter and the SV40 large T antigen, produced by Maroulakoou in 1994, developed prostate cancer. Mice that incorporated the C3 (1) promoter and the Polyoma middle T gene, produced by Tehranian in 1996, developed prostate cancer. In 1997, Zhang reported that mice with the C3 (1) promoter and the human be1-2 (human bcl-2) gene developed hyperplasia in the prostate but not cancer. Did not reach. Placenta G-Girl made by Perez-Stable in 1996 Mice incorporating the promoter of robin (fetal glova in) and the SV40 large T antigen gene developed prostate cancer. Kitsberg, which incorporated the MMTV promoter and KGF gene in 1996, produced hyperplasia in the prostate but no cancer. Animal models for cancer development are indispensable in elucidating the mechanisms of cancer development and in developing therapeutics for them.Model animals for various cancers have been created so far. Since the types of substances that can be tested vary depending on the substance, method, and animal species that induce the disease, it is desirable to establish as many animal models as possible. Rats, like mice, have a short pregnancy and sexual maturation period, have about 10 litters, and weigh about 10 times that of mice, so that sufficient materials are available for analysis. In addition, there are many useful points such as the accumulation of data on many chemical carcinogenesis. However, to date, no model rat has been established for prostate cancer development that can reliably generate prostate cancer and can be stably passaged. An object of the present invention is to provide a model rat for prostate cancer development that can generate prostate cancer including invasive cancer and can be stably subcultured. Disclosure of the invention
本発明者らは上記課題を解決するために鋭意研究し、 プロバシン遺伝 子のプロモー夕一の制御下にある S V 4 0ラージ T抗原の遺伝子をラッ 卜に導入することによって得られるトランスジエニックラッ トを継代し. 前立腺の病理組織の観察を行い、 前立腺癌を全例に発生する トランスジ エニックラッ ト系統を選択することにより、 前立腺癌を確実に発生する ことができ、 かつ安定的に継代することができる前立腺癌発生のモデル ラッ トを樹立できることを見い出し、 本発明を完成するに至った。
すなわち本発明は、 浸潤癌を含む前立腺癌を発生することができ、 安 定的に継代することができる前立腺癌発生のモデルラッ ト （請求項 1 ) や、 プロバシン遺伝子のプロモー夕一の制御下にある S V 4 0ラージ T 抗原の遺伝子が導入された請求項 1記載の前立腺癌発生のモデルラッ ト (請求項 2 ) や、 EcoRI と Ncol で切りだしたラッ トのプロバシン遺伝 子プロモーターの 5 ' —フランキング領域 （一 4 2 6〜十 3 2の 4 5 8 b p ) の下流に、 Ncolと BamHIで切りだした S V 4 0ラージ T抗原の 遺伝子 （ 5 1 6 4〜 2 6 7 6 ) を結合して得られる 2 9 44 b pからな る P B S V T ト ラ ンス ジー ンをス プラー グ一 ドー リ ー（Sprague- Dawley)系ラッ トの受精卵に導入し、 導入後の受精卵を仮親に移植し、 該仮親から得られる トランスジエニック雄ラッ トを野生型スプラーグ一 ドーリ一系雌ラッ トと交配することにより得られる請求項 1又は 2記載 の前立腺癌発生のモデルラッ ト （請求項 3 ) や、 前立腺癌発生のモデル ラッ トが、 P B S VTトランスジエニックラッ ト 2 9 7 1系統である請 求項 1〜 3のいずれか記載の前立腺癌発生のモデルラッ ト （請求項 4) に関する。 The present inventors have intensively studied to solve the above-mentioned problems, and have studied a transgenic rat obtained by introducing a gene for SV40 large T antigen under the control of the promoter of the probasin gene into a rat. By observing the pathological tissue of the prostate and selecting a transgenic rat strain that produces prostate cancer in all cases, prostate cancer can be reliably generated and passaged stably The present inventors have found that a model rat of prostate cancer occurrence that can be used can be established, and have completed the present invention. That is, the present invention provides a model rat for prostate cancer development (Claim 1) that can generate prostate cancer including invasive cancer and can be stably subcultured, and under the control of the promoter of the probasin gene. The gene for the SV40 large T antigen is introduced into the model rat for prostate cancer development according to claim 1 (claim 2), or the probasin gene promoter of the rat cut with EcoRI and Ncol is 5'- Downstream of the flanking region (1 426 to 1332 458 bp), the gene for SV40 large T antigen (5164 to 2676) cut out by Ncol and BamHI is linked. The 2944 bp PBSVT transgene obtained by the above procedure was introduced into fertilized eggs of Sprague-Dawley rats, and the fertilized eggs after introduction were transplanted into foster parents. The transgenic male rat obtained from the foster parent is wild-type Sprague-Dawley. 3. The model rat for prostate cancer development according to claim 1 or claim 2 (Claim 3) obtained by crossing with a Dory line female rat, or the model rat for prostate cancer development is PBS VT transgenic rat 2. 971 A model rat for the development of prostate cancer according to any one of claims 1 to 3, which is a one-line system (claim 4).
また本発明は、 EcoRI と Ncol で切りだしたラッ トのプロバシン遺伝 子プロモ一夕一の 5 ' —フランキング領域 （― 4 2 6〜十 3 2の 4 5 8 b p ) の下流に、 Ncolと BamHIで切りだした S V 4 0ラージ T抗原の 遺伝子 （ 5 1 6 4〜 2 6 7 6 ) を結合して得られる 2 9 44 b pからな る P B S V T ト ラ ンス ジー ンをス プラー グ一 ドー リ ー（Sprague- Dawley)系ラッ 卜の受精卵に導入し、 導入後の受精卵を仮親に移植し、 該仮親から得られる トランスジエニックラッ トを野生型スプラーグ一 ド ーリ一系ラッ トと交配し、 得られる トランスジエニックラッ トを以後同 様に継代し、 前立腺の病理組織の観察することにより、 前立腺癌を発生 する トランスジェニックラッ トを選択することを特徴とする、 浸潤癌を
含む前立腺癌を発生することができ、 安定的に継代することができる前 立腺癌発生のモデルラッ トの樹立方法 （請求項 5 ) や、 浸潤癌を含む前 立腺癌を発生することができ、 安定的に継代することができる前立腺癌 発生のモデルラッ トが、 P B S V Tトランスジェニックラッ ト 2 9 7 1 系統であることを特徴とする、 請求項 5記載の浸潤癌を含む前立腺癌を 発生することができ、 安定的に継代することができる前立腺癌発生のモ デルラッ 卜の樹立方法 （請求項 6 ) に関する。 In addition, the present invention provides a promoter for the probasin gene of a rat cut with EcoRI and Ncol, which is located at the 5'-flanking region (-425-bp 32-458 bp) of the promoter. A PBSVT transgene consisting of 2944 bp obtained by binding the SV40 large T antigen gene (5164-26676) cut out with BamHI was added to the plasmid. -(Sprague-Dawley) rats are introduced into a fertilized egg, the fertilized eggs after the introduction are transplanted into a foster parent, and the transgenic rat obtained from the foster parent is used as a wild-type Sprague-Dawley-type rat. After crossing, the obtained transgenic rat is similarly passaged thereafter, and by observing the histopathology of the prostate, a transgenic rat that produces prostate cancer is selected. A method for establishing a model rat for the development of prostate cancer that can generate stable prostate cancer and that can be stably passaged (Claim 5), and a method for generating prostate cancer including invasive cancer. The prostate cancer generation model including the invasive cancer according to claim 5, wherein the model rat model for prostate cancer generation that can be stably subcultured is the PBSVT transgenic rat line 2971. The present invention relates to a method for establishing a model rat for the development of prostate cancer that can be stably subcultured.
さらに本発明は、 前立腺癌発生前後の、 請求項 1〜 4のいずれか記載 の前立腺癌発生のモデルラッ トに被検物質を投与し、 該モデルラッ トに おける前立腺癌の発生及び Z又は進行の程度を測定 ·評価することを特 徴とする前立腺癌の発生及び Z若しくは進行促進物質又は発生及び Z若 しくは進行抑制物質のスクリーニング方法 （請求項 7 ) や、 前立腺癌発 生及び Z又は進行の程度の測定 · 評価が、 前立腺癌発生のモデルラッ ト から得られる前立腺癌の病理組織像の解析 · 評価であることを特徴とす る請求項 7記載の前立腺癌の発生及び Z若しくは進行促進物質又は発生 及び Z若しくは進行抑制物質のスクリーニング方法 （請求項 8 ) や、 前 立腺癌発生及び Z又は進行の程度の測定 · 評価が、 前立腺癌細胞におい て産生されるプロスタン酸ホスファタ一ゼ （P A P ) 及び Z又は前立腺 特異的抗原 （P S A ) の測定 · 評価であることを特徴とする請求項 7記 載の前立腺癌の発生及び/若しくは進行促進物質又は発生及び Z若しく は進行抑制物質のスクリーニング方法 （請求項 9 ) や、 前立腺癌発生及 び Z又は進行の程度を測定 · 評価するに際し、 前立腺癌発生のモデルラ ッ トと同種の野生型ラッ トの測定値と比較 · 評価することを特徴とする 請求項 7〜 9のいずれか記載の前立腺癌の発生及び Z若しくは進行促進 物質又は発生及び/若しくは進行抑制物質のスクリーニング方法 （請求 項 1 0 ) や、 請求項 7〜 1 0のいずれか記載の前立腺癌の発生及び/若
しくは進行促進物質又は発生及び Z若しくは進行抑制物質のスクリー二 ング方法により得られることを特徴とする前立腺癌の発生及び Z又は進 行促進物質 （請求項 1 1 ) や、 請求項 7〜 1 0のいずれか記載の前立腺 癌の発生及び Z若しくは進行促進物質又は発生及び/若しくは進行抑制 物質のスクリーニング方法により得られることを特徴とする前立腺癌の 発生及び Z又は進行抑制物質 （請求項 1 2 ) や、 前立腺癌の発生及び Z 又は進行抑制物質が、 前立腺癌に対する予防剤、 抑制剤若しくは治療剤 であることを特徴とする請求項 1 2記載の前立腺癌の発生及び Z又は進 行抑制物質 （請求項 1 3 ) や、 前立腺癌の発生及び 又は進行の抑制を 必要としている患者を治療するのに用いられる医薬組成物であって、 有 効成分として請求項 1 3記載の前立腺癌の発生及び/又は進行抑制物質 を含有することを特徴とする医薬組成物 （請求項 1 4 ) に関する。 図面の簡単な説明 Further, the present invention provides a method for administering a test substance to a model rat for prostate cancer development according to any one of claims 1 to 4 before and after the onset of prostate cancer, wherein the degree of the onset, Z or progression of prostate cancer in the model rat is measured. A method for screening prostate cancer occurrence and Z or progression promoting substances or Z or progression or progression inhibiting substances (Claim 7), and prostate cancer development and Z or progression of Z or progression. The prostate cancer development and Z or progression promoting substance according to claim 7, wherein the measurement and evaluation of the degree is analysis and evaluation of a histopathological image of prostate cancer obtained from a model rat of prostate cancer development. The method for screening for substances inhibiting the onset and progression of Z or progression (Claim 8) and the measurement and evaluation of the degree of onset and progression of prostate cancer and the progression of Z or progression are performed by prostasis produced in prostate cancer cells 8. The substance for promoting and / or promoting the development and / or progression of prostate cancer according to claim 7, which is used for measurement and evaluation of acid phosphatase (PAP) and Z or prostate specific antigen (PSA). Alternatively, when measuring and evaluating the degree of progression of prostate cancer and / or Z or progression, the measured value of a wild-type rat of the same type as the model rat of prostate cancer development is used in the screening method for a progression inhibitor (Claim 9). A method for screening prostate cancer occurrence and Z or progression promoting substances or occurrence and / or progression inhibiting substances (claim 10) according to any one of claims 7 to 9, characterized in that they are compared and evaluated. The occurrence of prostate cancer according to any one of 7 to 10 and / or Or a substance that promotes the development and progression of prostate cancer and is characterized by being obtained by a screening method of a substance that promotes progression or generation and Z or progression inhibiting substance (claim 11), and claims 7 to 1 0 The prostate cancer development and Z or progression inhibiting substance obtained by the screening method for prostate cancer development and Z or progression promoting substance or development and / or progression inhibiting substance according to any one of claims 0 to 12. The prostate cancer occurrence and Z or progress inhibitor according to claim 12, wherein the prostate cancer occurrence and Z or progress inhibitor is a prophylactic, inhibitor or therapeutic agent for prostate cancer. (Claim 13) or a pharmaceutical composition used for treating a patient in need of suppressing the development and / or progression of prostate cancer, wherein the pharmaceutical composition according to claim 13 is used as an active ingredient. Pharmaceutical composition characterized in that it contains a generator and / or progression inhibitor of standing adenocarcinoma related (claims 1-4). BRIEF DESCRIPTION OF THE FIGURES
第 1図は、 本発明の前立腺癌トランスジエニックラッ トモデル創出の ために用いた P B S V T遺伝子断片を示す図である。 発明を実施するための最良の形態 FIG. 1 is a diagram showing a PVSVT gene fragment used for creating a prostate cancer transgenic rat model of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION
本発明の前立腺癌発生のモデルラッ トとしては、 浸潤癌を含む前立腺 癌をほぼ全例に発生することができ、 かつ安定的に継代することができ るラッ ト系統であれば特に制限されるものではないが、 前立腺において 特異的に発現し、 アンドロゲンや亜鉛で調節される蛋白質をコードする 遺伝子であるプロバシン遺伝子のプロモーターの制御下に、 p 5 3蛋白 及び P b蛋白に直接結合してその機能を不活性化することによって癌を 誘発する S V 4 0ラージ T抗原の遺伝子を結合したトランスジーン （P B S V T トランスジーン） が導入されたトランスジエニックラッ 卜から
樹立された前立腺癌発生のモデルラッ ト、 例えば P B S VTトランスジ エニックラッ ト 2 9 7 L系統を具体的に挙げることができる。 The model rat of the present invention for the development of prostate cancer is particularly limited as long as it is a rat line that can generate prostate cancer including invasive cancer in almost all cases and can be passaged stably. Although not specifically regulated, it binds directly to p53 and Pb proteins under the control of the promoter of the probasin gene, a gene that is specifically expressed in the prostate and encodes a protein regulated by androgens and zinc. Induces cancer by inactivating functions. From a transgenic rat into which a transgene (PBSVT transgene) linked to the gene for SV40 large T antigen is introduced. An established model rat for the development of prostate cancer, for example, the PBS VT transgenic rat 297L strain can be specifically mentioned.
本発明の前立腺癌発生のモデルラッ トの樹立方法としては、 公知のト ランスジエニック動物の作製方法 （例えば、 Proc. Natl. Acad. Sci. USA 77:7380-7384,1980) を用いた方法を挙げることができ、 以下、 上記プ ロバシンのプロモーターの制御下に S V 4 0ラージ T抗原遺伝子が位置 する遺伝子断片をトランスジーンとして用いる場合を例にとって説明す る。 公知の方法に準じて調製した P B S VTトランスジーンを適当な発 現ベクターに入れて増幅し、 増幅後、 遺伝子断片 （P B S VT トランス ジーン） を切りだし、 かかる P B S VT トランスジーンをマイクロイン ジェクシヨ ン法によりラッ ト前核期受精卵へ導入し、 導入後の受精卵を 仮親に移植し産仔させる。 出生した仔ラッ 卜に P B S VTトランスジー ンが導入されているかどうかは、 体の一部 （例えば尾部先端） から DN Aを抽出し、 サザンプロッ ト分析や P C Rアツセィなどにより確認する ことができる。 P B S V Tトランスジーンの存在が確認された個体は初 代（Founder)であり、 この初代ラッ トと健常な野生型ラッ トを交配させ る。 得られた産仔 （F 1 ) の 5 0 %が P B S VTトランスジーンを承継 するカ^ P B S VTトランスジーンが導入されていることが確認された F 1 ラッ トを健常ラッ トと交配し、 以後同様に継代し、 前立腺の病理組 織の観察し、 前立腺癌を発生する トランスジエニックラッ トを選択する ことにより前立腺癌発生のモデルラッ ト系統を樹立することができる。 上記トランスジーンにおけるプロバシン遺伝子のプロモーターに代え て、 前立腺において特異的に発現する前立腺特異的抗原 （P S A) 又は 前立腺特異的膜抗原 （P S MA) に由来するプロモーターを用いること もできる。 癌を誘発する S V 4 0ラージ T抗原の遺伝子に代えて、 S V 4 0の温度感受性突然変異株 t s A 5 8のラージ T抗原遺伝子や、 アデ
ノウィルスの E 1 A遺伝子、 ヒ トパピ口一マウィルスの H P V 1 6遺伝 子等を用いることもできる。 また、 受精卵への導入方法として、 上記マ イク口インジクシヨ ン法に代えて、 静電パルス法、 リボソーム法、 リ ン 酸カルシウム法等も用いることができる。 As a method for establishing a model rat for the development of prostate cancer according to the present invention, a method using a known method for producing a transgenic animal (for example, Proc. Natl. Acad. Sci. USA 77: 7380-7384, 1980) is used. Hereinafter, a case where a gene fragment in which the SV40 large T antigen gene is located under the control of the above-mentioned probasin promoter is used as a transgene will be described as an example. A PBS VT transgene prepared according to a known method is put into an appropriate expression vector and amplified. After amplification, a gene fragment (PBS VT transgene) is cut out, and the PBS VT transgene is microinjected. Introduce to fertilized eggs in the pre-rat nucleus at the stage of transplantation, and transfer the fertilized eggs after the introduction to the foster mother to lay offspring. Whether the PBS VT transgene has been introduced into the offspring of a born rat can be confirmed by extracting DNA from a part of the body (for example, the end of the tail) and conducting Southern blot analysis or PCR assay. The individual in which the presence of the PBSVT transgene has been confirmed is the first generation (Founder), and this first generation rat is crossed with a healthy wild-type rat. 50% of the obtained offspring (F 1) were inherited from the PBS VT transgene, and it was confirmed that the PBS VT transgene had been introduced. Similarly, a model rat line for the development of prostate cancer can be established by observing the pathological tissue of the prostate and selecting a transgenic rat that develops prostate cancer. Instead of the probasin gene promoter in the above transgene, a promoter derived from a prostate specific antigen (PSA) or a prostate specific membrane antigen (PSMA) which is specifically expressed in the prostate can be used. Instead of the gene for the SV40 large T antigen that induces cancer, the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40, For example, the E1A gene of an influenza virus and the HPV16 gene of a human papilla virus can be used. In addition, as a method for introduction into a fertilized egg, an electrostatic pulse method, a ribosome method, a calcium phosphate method, or the like can be used in place of the above-mentioned mouth opening method.
また、 前記本発明の前立腺癌発生のモデルラッ トである P B S V T卜 ランスジエニックラッ ト 2 9 7 1系統は、 EcoRI と Ncol で切りだした ラッ トのプロバシン遺伝子プロモ一夕一の 5 ' —フランキング領域 （一 4 2 6〜+ 3 2の4 5 8 13 ） の下流に、 Ncolと BamHIで切りだした S V 4 0ラージ T抗原の遺伝子 （ 5 1 6 4〜 2 6 7 6 ) を結合して得ら れる 2 9 44 b pからなる P B S VTトランスジーンをスプラーグー ド 一リー（Sprague-Dawley)系ラッ 卜の受精卵に導入し、 導入後の受精卵を 仮親に移植し、 該仮親から得られる トランスジエニックラッ トを野生型 スプラ一グー ドーリー系ラッ 卜と交配し、 得られる トランスジエニック ラッ トを以後同様に継代し、 前立腺の病理組織の観察して、 前立腺癌を 発生する トランスジエニックラッ トを選択することにより、 浸潤癌を含 む前立腺癌を発生することができ、 安定的に継代することができる前立 腺癌発生のモデルラッ トとして樹立されたものである。 この P B S VT トランスジエニックラッ ト 2 9 7 1系統は、 名古屋巿立大学医学部第 1 病理学教室において継代されており、 一定の条件下で関係者は分譲を受 けることができる。 The PBSVT transgenic rat strain 2971, which is a model rat for the development of prostate cancer according to the present invention, is a 5′-flanking promoter of the rat probasin gene promoter cut with EcoRI and Ncol. Downstream of the region (1 4 2 6 to +3 2 4 5 8 13), the SV40 large T antigen gene (5 16 4 to 2 6 7 6) cut by Ncol and BamHI The resulting 2944 bp PBS VT transgene is introduced into fertilized eggs of a Sprague-Dawley rat, the fertilized eggs after the introduction are transplanted into a foster parent, and the transfection obtained from the foster parent is performed. The transgenic rat is crossed with a wild-type Sprague-Dawley rat, and the resulting transgenic rat is similarly passaged thereafter. By choosing a rat The invasive cancer can be generated including prostate cancer, which has been established as Moderura' capital of prostate cancer occurrence can be stably passaged. This PBSVT VT transgenic rat line 29771 has been passed on at the No. 1 Department of Pathology, Faculty of Medicine, Nagoya Ritsumeikan University, and under certain conditions, interested parties can receive a subdivision.
上記本発明の前立腺癌発生のモデルラッ トは、 前立腺癌発生のメカ二 ズム解明の他、 前立腺癌の発生 · 進行の促進物質又は抑制物質のスクリ 一二ング等に用いることができる。かかるスク リーニング方法としては、 前立腺癌発生前、 あるいは前立腺癌発生後に、 本発明の前立腺癌発生の モデルラッ トに被検物質を投与し、 該モデルラッ トにおける前立腺癌の 発生及び Z又は進行の程度を測定'評価する方法を挙げることができる。
また、 前立腺癌発生及び Z又は進行の程度の測定 ·評価方法としては、 被検物質を投与されたモデルラッ 卜から得られる前立腺癌の病理組織像 を解析 · 評価する方法を例示することができる。 そして、 前立腺癌発生 及び Z又は進行の程度を測定 ·評価するに際しては、 前立腺癌発生のモ デルラッ トと同種の野生型ラッ トを同時に用いることが、 個体レベルで 正確な比較実験をすることができることから好ましい。 The model rat for prostate cancer development according to the present invention can be used for elucidating the mechanism of prostate cancer development and for screening a substance that promotes or suppresses the development and progression of prostate cancer. As such a screening method, a test substance is administered to a model rat of prostate cancer development of the present invention before or after prostate cancer development, and the occurrence of prostate cancer and the degree of Z or progress in the model rat are determined. Measurement and evaluation methods can be mentioned. In addition, examples of the method for measuring and evaluating the degree of prostate cancer occurrence and Z or progression include a method for analyzing and evaluating a pathological image of prostate cancer obtained from a model rat to which a test substance has been administered. When measuring and evaluating the extent of prostate cancer development and Z or progression, it is necessary to use a model rat of prostate cancer development and a wild-type rat of the same species simultaneously, so that accurate comparison experiments can be performed at the individual level. It is preferable because it can be performed.
本発明のスクリーニング方法により得られる前立腺癌の発生及び Z又 は進行抑制物質は、 前立腺癌に対する予防剤、 抑制剤若しくは治療剤等 に用いることができる可能性がある。 例えば、 上記スクリーニング方法 において、 前立腺癌発生前の被検物質の投与は、 前立腺癌の予防薬剤の スクリーニングに適しており、 反対に前立腺癌発生後の被検物質の投与 は、 前立腺癌の治療剤 · 症状改善剤のスクリーニングに適している。 他 方、 前立腺癌の発生及び Z又は進行促進物質は、 前立腺癌の発生が不十 分なモデル動物において、 前立腺癌を充分に生起させたい場合などに有 用であると考えられる。 また、 本発明の前立腺癌の発生及びノ又は進行 の抑制を必要としている患者を治療するのに用いられる医薬組成物は、 有効成分としての前立腺癌の発生及び Z又は進行抑制物質と、 製剤化に 必要な公知の物質を含んでいる。 The substance that suppresses prostate cancer occurrence and Z or progression obtained by the screening method of the present invention may be used as a prophylactic, suppressive, or therapeutic agent for prostate cancer. For example, in the above screening method, administration of a test substance before the onset of prostate cancer is suitable for screening for a prophylactic cancer preventive agent, and conversely, administration of a test substance after the onset of prostate cancer is suitable for treatment of a prostate cancer therapeutic agent · Suitable for screening for symptom improvers. On the other hand, a substance that promotes the development of prostate cancer and promotes Z or progression is considered to be useful when it is desired to sufficiently generate prostate cancer in a model animal in which the development of prostate cancer is insufficient. Further, the pharmaceutical composition of the present invention used for treating a patient in need of suppressing the occurrence and progression of prostate cancer or the prostate cancer is characterized by comprising a substance inhibiting prostate cancer development and / or Z or progression as an active ingredient. Contains known substances required for
以下本発明を実施例に基づいて説明するが、 本発明はかかる実施例に より制限されるものではない。 Hereinafter, the present invention will be described based on examples, but the present invention is not limited to the examples.
( P B S V T トランスジーンの構築） (Construction of PBSTV transgene)
S V 4 0ラージ T抗原の遺伝子は、 ヒューマンサイエンス研究資源バ ンクから供与された、 pBluescriptllKS (-)のマルチクローニングサイ ト に Ncol— BamHI断片として組み込まれているものを使用した。 かかる pBluescriptllKS (-)から Ncolと BamHIで切りだした S V 4 0ラージ T 抗原の遺伝子 （ 5 1 6 4〜 2 6 7 6 ) を、 EcoRI と Ncol で切りだした
P ラッ トのプロバシン遺伝子プロモーターの 5 ' —フランキング領域 （― 4 2 6〜十 3 2の 4 5 8 b p ) の下流に結合し、 P B S VT トランスジ ーンを構築した （図 1 )。 この 2 9 4 4 b pからなる S V 4 0 ラージ T抗 原とスモール t抗原の両方を発現することができる P B S VT トランス ジーンは、 プラスミ ド pBluescriptll (トランスジーン社製） を用いて増 幅した後、 制限酵素 EcoRI と BamHIにより消化し、 1 %ァガ口一スゲ ル電気泳動を行い、 プラスミ ド部分を除去し、 直鎖状 DNA断片として 分離した。 分離した D N A断片を G E N E C L E A N E II ( BIO 101 INC社 ）を用いて回収し、 精製後に得られた D N Aを 5 fi /m 1 とな るように注入用バッファ一 （O.lmM EDTAを含む 10mM Tris-HCl， pH 7.6) に溶解し、 注入操作に使用するまで一 2 0 °Cで保存した。 As the gene for the SV40 large T antigen, a gene integrated into a multicloning site of pBluescriptllKS (-) as an Ncol-BamHI fragment, provided by the Human Science Research Resource Bank was used. From this pBluescriptllKS (-), the gene for SV40 large T antigen (5164 to 2676) cut with Ncol and BamHI was cut with EcoRI and Ncol. By binding to the downstream of the 5'-flanking region of the P-rat probasin gene promoter (-428 to 132, 458 bp), a PBS VT transgene was constructed (Fig. 1). The PBS VT transgene capable of expressing both the SV440 large T antigen and the small t antigen consisting of 294 bp was amplified using plasmid pBluescriptll (manufactured by Transgene). After digestion with the restriction enzymes EcoRI and BamHI, 1% agarose gel electrophoresis was performed to remove the plasmid portion and separated as a linear DNA fragment. The separated DNA fragments were recovered using GENECLEANE II (BIO101 INC), and the DNA obtained after purification was adjusted to 5 fi / m1 with an injection buffer (10 mM Tris-containing O.lmM EDTA). HCl, pH 7.6) and stored at 120 ° C until used for injection.
(トランスジエニックラッ 卜の作製） (Production of transgenic rat)
ラッ 卜の前核期受精卵への上記 P B S VT トランスジーン溶液のマイ クロインジェクショ ンは下記の要領で実施した。 8週齢のスプラ一グー ドーリー（SD， Sprague-Dawley)系雌ラッ トを明暗サイクル 1 2時間 （明 時間 4 : 0 0〜 ： L 6 : 0 0)、 温度約 2 3 °C、 湿度約 5 5 %で飼育し、 膣スメァ法により雌の性周期を観察してホルモン処理日を選択した。 雌 ラッ トに 1 5 0 I UZ k gの妊馬血清性性腺刺激ホルモン （日本全薬社 製 「P M Sゼンャク」） を腹腔内投与して過剰排卵処理を行い、 その 4 8 時間後に 7 5 I UZ k gのヒ ト絨毛性性腺刺激ホルモン （三共臓器社製 「プベロ一ゲン」） を投与した後、 雄との同居により交配を行わせ、 ヒ ト 絨毛性性腺刺激ホルモン投与 3 2時間後に卵管灌流により前核期受精卵 を採取した。卵管灌流及び卵の培養には mK R B液（Y. Yoshida and M.C. Chang , J. Reprod. Fertil., Vol.36, pp9-22(l974)) を使用した。 採取し た受精卵を 0. 1 %ヒアルロニダ一ゼ （シグマ社製） を含む mKR B液 中で 3 7 °C、 5分間の酵素処理を行い、 卵丘細胞を除去した後、 mKR
B液で 3回洗浄して酵素を除去し、 DNA注入操作まで C 02インキュ ベ—ター内 （c〇 25 %、 3 7 ：、 飽和湿度） に保存した。 この様にし て調製したウィスターラッ トの受精卵の雄性前核に、 前記 P B S VTト ランスジーン溶液を、受精卵 1個当たり 1 0分子となるように導入した。 導入した 2 0 0個の受精卵を 1 0匹の仮親に移植し、 4 0匹の産仔を得 た。 離乳直後の尾より調製した DNAを、 配列表配列番号 1及び 2記載 のプライマーを用いて P C R法により検定した。 その結果、 S D系の 4 匹の初代 （ F 0 ) の雄トランスジエニックラッ トを得た。 Microinjection of the PBS VT transgene solution into the pronuclear stage fertilized eggs of the rat was performed as follows. An 8-week-old Sprague-Dawley (Sprague-Dawley) female rat was subjected to a 12-hour light-dark cycle (bright time 4: 00-: L 6: 00), temperature about 23 ° C, humidity about The animals were bred at 55% and the hormonal cycle of the female was observed by the vaginal smear method, and the date of hormone treatment was selected. Superovulation was performed by intraperitoneally administering 150 IU kg of pregnant female serum gonadotropin (PMS Zenyaku, manufactured by Nippon Zenyaku Co., Ltd.) to female rats, and 48 hours later, 75 IUZ kg of human chorionic gonadotropin (Puberogen, manufactured by Sankyo Organ Co., Ltd.) was administered, and then mated with a male. The pronuclear stage fertilized eggs were collected by the method. For oviduct perfusion and egg culture, mK RB solution (Y. Yoshida and MC Chang, J. Reprod. Fertil., Vol. 36, pp9-22 (l974)) was used. The collected fertilized eggs were subjected to enzymatic treatment at 37 ° C for 5 minutes in mKR B solution containing 0.1% hyaluronidase (Sigma) to remove cumulus cells. The enzyme was removed by washing 3 times with solution B, to DNA injection operation C 0 2 incubator base - the coater (C_〇 2 5%, 3 7:, saturated humidity) and stored in. The PBS VT transgene solution was introduced into the male pronucleus of the fertilized eggs of Wistar rat thus prepared so that the number of molecules per fertilized egg was 10 molecules. The introduced 200 fertilized eggs were transplanted into 10 foster parents to obtain 40 offspring. DNA prepared from the tail immediately after weaning was assayed by the PCR method using the primers shown in SEQ ID NOs: 1 and 2 in the Sequence Listing. As a result, four primary (F 0) male transgenic rats of the SD strain were obtained.
かかる初代の雄トランスジェニックラッ トを健常な野生型 S D系雌ラ ッ トと交配して雑種第 1代 （F 1 ) を作製して、 経時的に全身臓器にお ける病変について検討した。 4匹のトランスジエニックラッ ト中 2 9 4 4系統及び 2 9 7 1系統の 2匹がライン化可能であった。 2 9 7 1系は 安定的な継代が可能であり、 P B S VTトランスジエニックラッ トとし て樹立することができた。 一方、 2 9 44系は継代を重ねるにつれ水頭 症の出現が高頻度となり系が途絶してしまった。 The first male transgenic rat was crossed with a healthy wild-type SD female rat to produce the first hybrid (F1), and the lesions in systemic organs were examined over time. Two out of four transgenic rats, 2 944 4 lines and 2 971 lines, could be made into lines. Line 29771 was able to be stably passaged and established as a PBS VT transgenic rat. On the other hand, in the 2944 strain, the occurrence of hydrocephalus increased with the passage of time, and the strain was interrupted.
2 9 44系統及び 2 9 7 1系統の F 0は、 1 2週齢頃より両下肢麻痺 が出現し 1 4週及び 2 5週に死亡した。 両者とも前立腺腹葉でびまん性 に高度異型過形成が認められたが、 癌病巣は観察されなかった。 S V 4 0 T抗原は前立腺全葉に発現していたが、 特に腹葉で高度に発現してい た。 また両者とも脊髄腫瘍、 さらに 2 9 7 1系統では舌下部腫瘍も認め られ、 いずれも悪性小円形細胞腫瘍で S V 4 0 T抗原陽性であった。 F0 of the 2944 strain and the 2971 strain developed bilateral lower limb paralysis from around the age of 12 weeks and died at 14 and 25 weeks. In both cases, diffuse atypical hyperplasia was observed in the ventral lobe of the prostate, but no cancer lesion was observed. The SV40 T antigen was expressed on all lobes of the prostate gland, but was highly expressed especially on the abdominal lobe. In both cases, spinal cord tumors were observed, and in addition, sublingual tumors were observed in the 2971 line, and both were malignant small round cell tumors and were positive for SV40 T antigen.
2 9 7 1系及び 2 9 44系の継代において、 死亡したもの及び屠殺し たものに関する全体的な所見を表 1 「 P B S VTトランスジエニックラ ッ ト 2 9 7 1系の全体的な所見 （死亡又は屠殺の場合）」 及び表 2 「P B S V T トランスジェニックラッ ト 2 9 44系の全体的な所見 （死亡又は 屠殺の場合）」 に示す。 また、 それらの前立腺の病理組織学的知見を第 3
表「 P B S VT トランスジエニックラッ 卜の前立腺の病理組織学的知見」 に示す。 表 1に示されるように、 2 9 7 1系ラッ トは、 継代可能なこと が確認された。 前立腺以外では舌、 脊髄周辺に小細胞性未分化悪性腫瘍 の発生がみられた。 また、 表 2に示されるように、 2 9 44系ラッ トに は、 水頭症で死亡する個体が多かった。 前立腺以外では舌、 脊髄周辺、 その他に小細胞性未分化悪性腫瘍の発生がみられた。 表 1 Table 1 `` Overall findings of the PBS VT transgenic rat line 2971 '' for the deaths and slaughter at passages 2971 and 2944 (In case of death or slaughter) "and Table 2" Overall findings of PBSVT transgenic rat line 2944 (in case of death or slaughter) ". In addition, histopathological findings of those prostates were It is shown in the table “Histopathological findings of the prostate in PBS VT transgenic rats”. As shown in Table 1, it was confirmed that the 2971 rat could be subcultured. Except for the prostate, small cell undifferentiated malignant tumors were found around the tongue and spinal cord. In addition, as shown in Table 2, many individuals in the 2944 series rat died of hydrocephalus. Apart from the prostate, small cell undifferentiated malignant tumors were found in the tongue, around the spinal cord, and elsewhere. table 1
PBSVTトランスジエニックラット 2971系の全体的な所見 （死亡又は屠殺の場合） 動物番号 性別 週齢 死亡又は屠殺 全体的所見 Overall findings of PBSVT transgenic rat strain 2971 (if dead or sacrificed) Animal number Gender Week age Dead or sacrificed Overall findings
F0 M 25 死亡 脊髄、 舌及びリンパ節の腫瘍 F0 M 25 dead Tumor of spinal cord, tongue and lymph nodes
27 F 26 死亡 脊髄、 舌及びリンパ節の腫瘍 27 F 26 dead Tumor of spinal cord, tongue and lymph nodes
28 M 3 屠殺 28 M 3 slaughtered
31 F 26 死亡 脊髄、 舌及びリ ンパ節の腫瘍 31 F 26 dead Tumor of spinal cord, tongue and lymph nodes
34 F 26 屠殺 脊髄、 舌及びリンパ節の腫瘍 34 F 26 Sacrifice Tumor of spinal cord, tongue and lymph nodes
55 M 17 死亡 脊髄の腫瘍 55 M 17 dead Spinal cord tumor
56 M 22 死亡 脊髄、 舌及びリンパ節の腫瘍 56 M 22 dead Tumor of spinal cord, tongue and lymph nodes
59 M 24 屠殺 脊髄、 舌及びリ ンパ節の腫瘍 59 M 24 Sacrifice Tumor of spinal cord, tongue and lymph nodes
61 M 15 死亡 脊髄、 舌及びリ ンパ節の腫瘍 61 M 15 dead Tumor of spinal cord, tongue and lymph nodes
62 M 16 死亡 脊髄、 舌及びリンパ節の腫瘍 62 M 16 dead Tumor of spinal cord, tongue and lymph nodes
66 F 19 死亡 脊髄、 舌及びリ ンパ節の腫瘍 66 F 19 dead Tumor of spinal cord, tongue and lymph nodes
69 F 13 死亡 舌及びリ ンパ節の腫瘍 69 F 13 dead Tumor of tongue and lymph nodes
F1 70 F 25 屠殺 脊髄、 舌及びリンパ節の腫瘍 F1 70 F25 Sacrifice Spinal cord, tongue and lymph node tumors
75 M 24 死亡 脊髄、 舌及びリンパ節の腫瘍 75 M 24 dead Tumor of spinal cord, tongue and lymph nodes
76 M 19 死亡 脊髄、 舌及びリ ンパ節の腫瘍 76 M 19 dead Tumor of spinal cord, tongue and lymph nodes
77 F 21 死亡 脊髄、 舌及びリ ンパ節の腫瘍 77 F 21 dead Tumor of spinal cord, tongue and lymph nodes
78 F 24 死亡 脊髄、 舌及びリンパ節の腫瘍 78 F 24 dead Tumor of spinal cord, tongue and lymph nodes
79 F 24 死亡 舌及びり ンパ節の腫瘍 79 F 24 dead Tumor on tongue and lymph nodes
81 F 25 屠殺 脊髄、 舌及びリ ンパ節の腫瘍 81 F 25 Sacrifice Tumor of spinal cord, tongue and lymph nodes
84 25 死亡 舌及びリ ンパ節の腫瘍 84 25 Death Tongue and lymph node tumor
100 F 21 死亡 脊髄、 舌及びリンパ節の腫瘍 100 F 21 dead Tumor of spinal cord, tongue and lymph nodes
102 M 17 死亡 脊髄、 舌及びリ ンパ節の腫瘍 102 M 17 dead Tumor of spinal cord, tongue and lymph nodes
104 M 15 死亡 脊髄、 舌及びリ ンパ節の腫瘍 104 M 15 dead Tumor of spinal cord, tongue and lymph nodes
105 M 20 屠殺
表 2 105 M 20 slaughtered Table 2
P B SVTトランスジエニックラット 2944系の全体的な所見 （死亡又は屠殺の場合）
Overall findings of PB SVT transgenic rat strain 2944 (in case of death or slaughter)
表 3 Table 3
PBSVT卜ランスジエニックラッ卜の前立腺の病理組織学的知見 Histopathological findings of the prostate in PBSVT transgenic rats
AH： 異型過形成 CA： 癌 n. p. ： テス 卜せず
2 9 7 1系の 1 5週齢の P B S VTトランスジエニックラッ ト 5匹を 屠殺して、 前立腺及び精巣を病理組織学的に調べた。 その結果、 前立腺 各葉でほぼ全例に腺癌の発生がみられ、 これらは腺構造中に配置された 多くの有糸分裂像をもつ異型癌細胞であった。 特に前葉では浸潤癌の発 生が観察された。 結果を表 4 「 P B S VTトランスジエニックラッ トの 前立腺及び精巣の病理組織学的知見」 に示す。 表 4 AH: Atypical hyperplasia CA: Cancer np: Not tested Five 15-week-old PBS VT transgenic rats of line 2971 were sacrificed and the prostate and testes were examined histopathologically. As a result, almost all cases of adenocarcinoma were found in each lobe of the prostate gland, and these were atypical cancer cells with many mitotic figures arranged in glandular structures. In particular, the occurrence of invasive cancer was observed in the anterior lobe. The results are shown in Table 4 “Histopathological findings of prostate and testis in PBS VT transgenic rat”. Table 4
P B S VT卜ランスジエニックラッ卜の前立腺及び精巣の病理組織学的知見 Histopathological findings of prostate and testis in PBS VT transgenic rats
AH： 異型過形成 Ca： 癌 0： 浸潤癌 AH: Atypical hyperplasia Ca: Cancer 0: Invasive cancer
P B S VTトランスジエニックラッ トにおける S V 4 0ラ一ジ T抗原 のタンパク発現を免疫組織化学的に観察した結果、 前立腺各葉、 唾液腺 導管及び精巣セルトリ細胞に認められた。 その結果を表 5 「P B S VT トランスジエニックラッ トにおける S V 4 0 T抗原のタンパク発現の免 疫組織化学的観察結果」 に示す。
As a result of immunohistochemical observation of protein expression of SV40 radioactive T antigen in PBS VT transgenic rat, it was observed in each lobe of prostate, salivary gland duct, and testicular Sertoli cells. The results are shown in Table 5 “Immunohistochemical observations of SV40 T antigen protein expression in PBS VT transgenic rats”.
表 5 Table 5
P B S V T卜ランスジエニックラツ卜における P B S V T
S V 4 0 T抗原のタンパク発現の免疫組織ィヒ学的観察結果 Immunohistochemical observation of protein expression of SV40 T antigen
前立腺 唾液腺 Prostate salivary gland
複葉 + + 導管細胞 土 Compound leaf + + duct cell soil
背葉 + + 腺房細胞 Dorsal lobe + + acinar cells
側葉 + + 結腸 Lateral lobe + + colon
前葉 + 肝臓 一 Anterior lobe + liver
精巣 一 薛臓 Testes
睾丸 胸腺 一 Testis thymus
セル卜リ細胞 土 リンパ節 Sertri cells soil lymph nodes
副睾丸 大脳 Epididymis cerebrum
肺 小脳 Lung cerebellum
+ + ： 強い要請 + ： 中程度の陽性 + +: Strong request +: Moderate positive
土 ： 弱い陽性 - ： 陰性 Sat: Weak positive-: Negative
テストステロンの投与による癌発生への影響を調べる次の実験を行 た。 The following experiment was conducted to examine the effects of testosterone administration on cancer development.
雄の P B S V Tトランスジエニックラッ トを 5週齢に 3群に分け、 第 1群は対照群とし、 第 2群は精巣摘出、 第 3群は 1 . 5 cm シリコンチ ユーブ内に充填したテス トステロンを皮下に移植し、 1 5週齢で全群剖 検屠殺し、 前立腺の病変を病理組織学的に検索した。 第 1群の対照群で は全例に前立腺前葉にびまん性に高度異型過形成がみられ、 一部では癌 の発生を認めた。 第 2群では前立腺は全体に萎縮しており腫瘍性病変は 認められなかった。 第 3群では前立腺全体が著明に腫大しており、 浸潤 癌の発生を高率に認めた。 しかし、 テストステロンを 1 5週投与した後 で去勢すると前立腺は退縮し、 すべての増殖性病巣は消失する。 これら の所見より、 この P B S V T トランスジエニックラッ 卜の前立腺腫瘍発 生はアンドロゲン依存性であり、 テストステロンにより短期間かつ顕著 に前立腺腫瘍を進展させることが明かとなった。 さらに、 5 3及び？
bを不活性化することによって前立腺癌が発生することが立証された。 産業上の利用可能性 Male PBSVT transgenic rats were divided into three groups at the age of 5 weeks, the first group was used as a control group, the second group was orchidectomized, and the third group was testosterone filled in a 1.5 cm silicon tube. Subcutaneously transplanted, all groups were sacrificed at the age of 15 weeks by autopsy, and prostate lesions were examined histopathologically. In the first group of controls, all patients had diffuse high-grade atypical hyperplasia in the anterior lobe of the prostate gland, and some developed cancer. In the second group, the prostate was completely atrophied and no neoplastic lesions were observed. In group 3, the entire prostate was markedly swollen, and the incidence of invasive cancer was high. However, castration after 15 weeks of testosterone administration causes the prostate to regress and all proliferative lesions to disappear. These findings revealed that the prostate tumor development of this PBSVT transgenic rat was androgen-dependent, and that testosterone significantly and rapidly developed prostate tumors. In addition, 53 and? It has been demonstrated that inactivating b produces prostate cancer. Industrial applicability
この動物モデルは前立腺癌を早期かつ高率に惹起させることが可能で ある。 したがって、 前立腺発癌過程を詳細に解析したり、 前立腺癌の治 療剤を開発するための前立腺癌モデルとして極めて有用である。 本発明 トランスジエニックラッ トは、 その形質より医学研究分野において有用 である。
This animal model can cause prostate cancer early and at a high rate. Therefore, it is extremely useful as a prostate cancer model for analyzing the prostate carcinogenesis process in detail and developing therapeutic agents for prostate cancer. The transgenic rat of the present invention is useful in the medical research field due to its trait.
Claims
1. 浸潤癌を含む前立腺癌を発生することができ、 安定的に継代するこ とができる前立腺癌発生のモデルラッ ト。 1. A model rat for the development of prostate cancer that can generate prostate cancer, including invasive cancer, and can be passaged stably.
2. プロバシン遺伝子のプロモーターの制御下にある S V 4 0ラージ T 抗原の遺伝子が導入された請求項 1記載の前立腺癌発生のモデルラッ ト, 2. The model rat for the development of prostate cancer according to claim 1, wherein the gene for SV40 large T antigen under the control of the promoter of the probasin gene has been introduced,
3. EcoRI と Ncol で切りだしたラッ トのプロバシン遺伝子プロモー夕 —の 5 ' —フランキング領域 （― 4 2 6〜十 3 2の 4 5 8 b p) の下流 に、 Ncolと BamHIで切りだした S V 4 0ラージ T抗原の遺伝子 （ 5 1 64〜 2 6 7 6 ) を結合して得られる 2 9 4 4 b pからなる P B S VT トランスジーンをスプラーグ一 ドーリ一（Sprague-Dawley)系ラッ トの 受精卵に導入し、 導入後の受精卵を仮親に移植し、 該仮親から得られる トランスジエニック雄ラッ トを野生型スプラーグ一 ドーリー系雌ラッ ト と交配することにより得られる請求項 1又は 2記載の前立腺癌発生のモ デノレラッ 卜。 3. Provacin gene promoter of rat cut with EcoRI and Ncol — 5 ′ — Flanking region (-428 to 1332, 458 bp), cut with Ncol and BamHI Fertilization of Sprague-Dawley rat with PBS VT transgene consisting of 244 bp obtained by binding SV40 large T antigen gene (5164-26676) The transgenic male rat obtained by introducing the fertilized egg into an egg, transplanting the fertilized egg after the introduction into a foster parent, and crossing the transgenic male rat obtained from the foster egg with a wild-type Sprague-Dawley female rat. Moderate study of prostate cancer development.
4. 前立腺癌発生のモデルラッ 卜が、 P B S VT トランスジエニックラ ッ ト 2 9 7 1系統である請求項 1〜 3のいずれか記載の前立腺癌発生の モ " Jレラッ 卜。 4. The model for prostate cancer development according to any one of claims 1 to 3, wherein the model rat for the development of prostate cancer is the PBST VT transgenic rat line 2971.
5. EcoRI と Ncol で切りだしたラッ 卜のプロバシン遺伝子プ口モータ —の 5 ' —フランキング領域 （― 4 2 6〜十 3 2の 4 5 8 b p) の下流 に、 Ncolと BamHIで切りだした S V 4 0ラージ T抗原の遺伝子 （ 5 1 6 4〜 2 6 7 6 ) を結合して得られる 2 9 44 b pからなる P B S VT トランスジーンをスプラーグ一 ドーリ一（Sprague-Dawley)系ラッ トの 受精卵に導入し、 導入後の受精卵を仮親に移植し、 該仮親から得られる トランスジエニックラッ トを野生型スプラーグ一 ドーリ一系ラッ トと交 配し、 得られる トランスジエニックラッ トを以後同様に継代し、 前立腺
の病理組織の観察することにより、 前立腺癌を発生する トランスジェニ ックラッ 卜を選択することを特徴とする、 浸潤癌を含む前立腺癌を発生 することができ、 安定的に継代することができる前立腺癌発生のモデル ラッ トの樹立方法。 5. The 5'-flanking region of the rat probasin gene motor cut out by EcoRI and Ncol is cut by Ncol and BamHI downstream of the flanking region (-428 to 1332, 458 bp). The PBS VT transgene of 2944 bp obtained by binding the SV40 large T antigen gene (5164 to 2676) obtained from the Sprague-Dawley rat The transgenic rat is introduced into a fertilized egg, the fertilized egg after the introduction is transplanted into a foster parent, and the transgenic rat obtained from the foster parent is bred with a wild-type Sprague-Dawley rat, and the resulting transgenic rat is obtained. After that, the same procedure was performed, and the prostate Prostate cancer including invasive carcinoma, characterized by selecting a transgenic rat that generates prostate cancer by observing the histopathology of the prostate. How to establish a model rat for cancer development.
6 . 浸潤癌を含む前立腺癌を発生することができ、 安定的に継代するこ とができる前立腺癌発生のモデルラッ ト力 、 P B S V T トランスジェニ ックラッ ト 2 9 7 1系統であることを特徴とする、 請求項 5記載の浸潤 癌を含む前立腺癌を発生することができ、 安定的に継代することができ る前立腺癌発生のモデルラッ トの樹立方法。 6. Prosthetic carcinoma including invasive carcinoma can be generated and can be stably passaged. It is a model strain of PBSVT transgenic rat. A method for establishing a model rat for prostate cancer development, which is capable of generating prostate cancer including invasive cancer according to claim 5, and capable of stable passage.
7 . 前立腺癌発生前後の、 請求項 1 〜 4のいずれか記載の前立腺癌発生 のモデルラッ トに被検物質を投与し、 該モデルラッ トにおける前立腺癌 の発生及び Z又は進行の程度を測定 · 評価することを特徴とする前立腺 癌の発生及び Z若しくは進行促進物質又は発生及び Z若しくは進行抑制 物質のスクリーニング方法。 7. A test substance is administered to the model rat for prostate cancer development according to any one of claims 1 to 4 before and after the onset of prostate cancer, and the degree of prostate cancer occurrence, Z or progress in the model rat is measured and evaluated. A method for screening a substance for promoting and / or inhibiting Z or progression of prostate cancer.
8 . 前立腺癌発生及び Z又は進行の程度の測定 · 評価が、 前立腺癌発生 のモデルラッ 卜から得られる前立腺癌の病理組織像の解析 · 評価である ことを特徴とする請求項 7記載の前立腺癌の発生及び Z若しくは進行促 進物質又は発生及び Z若しくは進行抑制物質のスクリーニング方法。8. The prostate cancer according to claim 7, wherein the measurement / evaluation of prostate cancer occurrence and the extent of Z or progression is an analysis / evaluation of a histopathological image of prostate cancer obtained from a model rat of prostate cancer occurrence. A screening method for the occurrence and Z or progression promoting substance or the occurrence and Z or progression inhibiting substance.
9 . 前立腺癌発生及び Z又は進行の程度の測定 ' 評価が、 前立腺癌細胞 において産生されるプロスタン酸ホスファターゼ （P A P ) 及び/又は 前立腺特異的抗原 （ P S A ) の測定 ' 評価であることを特徴とする請求 項 7記載の前立腺癌の発生及び Z若しくは進行促進物質又は発生及び/ 若しくは進行抑制物質のスクリーニング方法。 9. The measurement of prostate cancer development and the extent of Z or progression 'evaluation is the measurement of prostanoate phosphatase (PAP) and / or prostate-specific antigen (PSA) produced in prostate cancer cells. 8. The method for screening for a substance for promoting and / or progression of prostate cancer or a substance for suppressing the occurrence and / or progression of prostate cancer according to claim 7.
1 0 . 前立腺癌発生及び/又は進行の程度を測定 ' 評価するに際し、 前 立腺癌発生のモデルラッ トと同種の野生型ラッ トの測定値と比較 · 評価 することを特徴とする請求項 7 〜 9のいずれか記載の前立腺癌の発生及
びノ若しくは進行促進物質义は発生及びノ若しくは進行抑制物質のスク リーニング方法。 10. The method according to claim 7, wherein the degree of prostate cancer development and / or progression is measured and evaluated by comparing / evaluating with a measurement value of a wild type rat of the same kind as a model rat of prostate cancer development. Occurrence of prostate cancer according to any one of to 9 ノ or 発 生 or 义 発 生。。 発 生 発 生 方法 方法 方法.
1 1 . 請求項 7〜 1 0のいずれか記載の前立腺癌の発生及び Z若しくは 進行促進物質又は発生及び/若しくは進行抑制物質のスクリ一ニング方 法により得られることを特徴とする前立腺癌の発生及び Z又は進行促進 物質。 11. Prostate cancer development according to any one of claims 7 to 10, and prostate cancer characterized by being obtained by a screening method of Z or a progression promoting substance or a developmental and / or progression inhibiting substance. And Z or a progress promoting substance.
1 2 . 請求項 7〜 1 0のいずれか記載の前立腺癌の発生及びノ若しくは 進行促進物質又は発生及び Z若しくは進行抑制物質のスクリーニング方 法により得られることを特徴とする前立腺癌の発生及び Z又は進行抑制 物質。 12. Prostate cancer onset and Z obtained by the method for screening for prostate cancer onset and progression or progression or Z and progression inhibitory substances according to any one of claims 7 to 10. Or a progress inhibiting substance.
1 3 . 前立腺癌の発生及び Z又は進行抑制物質が、 前立腺癌に対する予 防剤、 抑制剤若しくは治療剤であることを特徴とする請求項 1 2記載の 前立腺癌の発生及びノ又は進行抑制物質。 13. The substance according to claim 12, wherein the substance inhibiting the occurrence and progress of prostate cancer or Z or progression is a prophylactic, inhibitor or therapeutic agent for prostate cancer. .
1 4 . 前立腺癌の発生及び Z又は進行の抑制を必要としている患者を治 療するのに用いられる医薬組成物であって、 有効成分として請求項 1 3 記載の前立腺癌の発生及び Z又は進行抑制物質を含有することを特徴と する医薬組成物。
14. A pharmaceutical composition used to treat a patient in need of suppressing the occurrence and progression of Z or progression of prostate cancer, wherein the onset or Z or progression of prostate cancer according to claim 13 is used as an active ingredient. A pharmaceutical composition characterized by containing an inhibitor.
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| AU2000251092A AU2000251092A1 (en) | 2000-02-21 | 2000-06-13 | Mdel rats with the onset of prostatic cancer |
| CA002400966A CA2400966C (en) | 2000-02-21 | 2000-06-13 | Rat model with the onset of prostate cancer |
| US10/204,504 US7105717B1 (en) | 2000-02-21 | 2000-06-13 | Model rat with the onset of prostatic cancer |
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| JP2000042491A JP2001231402A (en) | 2000-02-21 | 2000-02-21 | Prostatic cancer developed model rat |
| JP2000-42491 | 2000-02-21 |
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Non-Patent Citations (8)
| Title |
|---|
| A.D. Borowsky et al., Laboratory Investigation, vol. 76, no. 1, page 70A (1997). * |
| B. Charreau et al., Transgenic Research, vol. 5, pages 223-234 (1996). * |
| J.E. Green et al., Prostate, vol. 36, pages 59-63 (1998). * |
| S. Hochi et al., Animal Biotechnology, vol. 1, no. 2, pages 175-184 (1990). * |
| S. Kasper et al., Laboratory Investigation, vol. 78, no. 3, pages 319-333 (1998). * |
| S.V. Garde et al., Cancer Letters, vol. 70, pages 159-166 (1993). * |
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| T. Takahashi et al., Nippon Gan Gakkai Soukai Kiji, Dai 58 kai, Soukai (Hiroshima), 29 September - 01 October 1999 (30 November 1999), page 406. * |
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| CA2400966C (en) | 2009-05-19 |
| CA2400966A1 (en) | 2001-08-23 |
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